1. Field of the Invention
The present invention relates to a trehalose-releasing enzyme, and its preparation and uses, more particularly, to a novel trehalose-releasing enzyme which specifically hydrolyzes the linkage between a trehalose moiety and the remaining glycosyl moiety in non-reducing saccharides having a trehalose structure as an end unit and having a glucose polymerization degree of 3 or higher, and to the preparation of the enzyme. The present invention further relates to microorganisms capable of form the enzyme, trehalose obtained by us the enzyme, and compositions contain the trehalose.
2. Description of the Prior Art
Trehalose or .alpha., .alpha.-trehalose has been known as a non-reducing saccharide consisting of Glucose units. As is described in Advances in Carbohydrate Chemistry, Vol. 18, pp. 201-225 (1963), published by Academic Press, USA, and Applied and Environmental Microbiology, Vol. 56, pp. 3,213-3,215 (1990), trehalose widely exists in microorganisms, mushrooms, insects, etc., though the content is relatively low. Trehalose is a non-reducing saccharide, so that it neither reacts with substances containing amino groups such as amino acids and proteins, induces the amino-carbonyl reaction, nor deteriorates amino acid-containing substances. Thus, trehalose would be used without a fear of causing an unsatisfactory browning and deterioration. Because of these, the establishment of the industrial-scale preparation of trehalose has been in great demand.
Conventional preparations of trehalose are, for example, those which are disclosed in Japanese Patent Laid-Open No. 154,485/75 wherein microorganisms are utilized, and reported in Japanese Patent Laid-Open No. 216,695/83 wherein maltose is converted into trehalose by using maltose- and trehalose-phosphorylases in combination. The former, however, is not suitable for the industrial-scale preparation because the content of trehalose present in microorganisms used as a starting material is usually lower than 15 w/w % (the wording "w/w %" is abbreviated as "%" in the specification, unless otherwise specified), on a dry solid basis (d.s.b.), and the extraction and purification steps are complicated. The latter has the following demerits: Since trehalose is formed via glucose-1-phosphate, the concentration of maltose as a substrate could not be set to a satisfactorily high-level; (ii) the enzymatic reaction systems of the phosphorylases are reversible reactions, and their yields of the objective trehalose are relatively low; and (iii) it is substantially difficult to retain their reaction systems stably and to continue their enzymatic reactions smoothly. Thus, these conventional preparations have not been actually used as an industrial-scale preparation.
As regards the preparation of trehalose, it is reported in the column titled "Oligosaccharides" in the chapter titled "Current Status of Starch Application Development and Related Problems" in "Food Chemicals", No. 88, pp.67-72 (August, 1992) that "In spite of a wide applicability of trehalose, the enzymatic preparation via a direct saccharide-transfer reaction or a hydrolytic reaction has been reported to be scientifically almost impossible in this field." Thus, the preparation of trehalose by an enzymatic reaction using starch as a material has ben deemed to be scientifically very difficult.
It is known that partial starch hydrolysates, prepared from a material starch such as liquefied starch, dextrins and maltooligosaccharides, exhibit a reducing power owing to their reducing end groups. The reducing power of these reducing partial starch hydrolysates is generally expressed by "Dextrose Equivalent (DE) value", based on a dry weight. It is known that among reducing partial starch hydrolysates those with a relatively-high DE value generally have a relatively-low molecular weight and viscosity, as well as a relatively-high level of sweetness and reactivity, and readily react with substances having amino groups such as amino acids and proteins to cause an unsatisfactory browning, smell and deterioration of their quality.
Since the properties of reducing partial starch hydrolysates are varied dependently on their DE values, the relationship between reducing partial starch hydrolysates and their DE values is significant. It has been even believed impossible to break away the relationship in this field.
The present inventors, however, did change this common sense and succeeded to establish a preparation of trehalose as disclosed in Japanese Patent Application No. 362,131/92 wherein trehalose is directly produced from non-reducing partial starch hydrolysates by allowing glucoamylase together with a non-reducing saccharide-forming enzyme capable of forming non-reducing saccharides, having a trehalose structure as an end unit and having a degree of glucose polymerization of 3 or higher, to act on reducing partial starch hydrolysates having a degree of glucose polymerization of 3 or higher, prepared from a material starch. Although the preparation of trehalose yields trehalose from non-reducing partial starch hydrolysates in a yield of about 30% and can be feasible as an industrial-scale preparation, there still remains some fear of resulting in a high production cost in view of the trehalose yield. Therefore, the establishment of a novel preparation of trehalose, which forms trehalose from non-reducing partial starch hydrolysates in an increased yield, is in great demand.